Sensitive detection of K-ras mutations augments diagnosis of colorectal cancer metastases in the liver

Postoperative survival of colorectal cancer patients is often delineated by metastases spreading to the liver. Current clinical diagnostic procedures are unable to discover micrometastases in this organ. Our aim was to develo p a diagnostic tool for detecting micrometastases that are present at the t ime of surgery. Therefore, a PCR-RFLP assay was set up tracking point mutat ions of the K-ras oncogene at codons 12 and 13, based on mismatch primers a nd restriction enzymes BstXI and XcmI. The detection limit of this assay wa s one mutant in one million wild-type cells. One hundred forty-two patients with colorectal carcinoma were screened for these mutations in tissue samp les from their tumor, proximally adjacent mucosa, and liver. Of these, 67 p atients (46%) were positive for a K-ras mutation, of which 58 had codon 12 and 9 had codon 13 mutations. No patient without a K-ras-positive tumor sho wed a mutation in mucosa, but 11 patients with a K-ras-positive tumor (11 o f 58; 19%) were found to bear a K-ras mutation in their mucosa, and in 21 p atients (21 of 64; 33%), a K-ras mutation was detected in liver tissue. Seq uencing of all mutated samples revealed a 92% confirmation of PCR-RFLP resu lts. In summary, the assay is a useful tool for detecting K-ras codon 12 an d 13 mutations and allows early proof of molecular determinants of liver me tastases. Such knowledge will improve the staging of colorectal cancer pati ents and could beneficially influence their prognosis if followed by an eff ective therapy.